Journal of Insect Physiology

Seasonal variation in day length provides a reliable cue that allows insects to anticipate upcoming environmental challenges. Here, we demonstrate that photoperiod induces pronounced, sex-specific immune priming in the linden bug Pyrrhocoris apterus. Females exposed to short-day, diapause-inducing conditions exhibited broadly enhanced immune activity compared with long-day females, whereas immune parameters in males were largely unaffected by photoperiod. Short-day females showed increased immune cell abundance, elevated expression of immune-related genes, enhanced humoral immune activity, and increased resistance to bacterial infection. Importantly, photoperiod-induced immune priming depended on a functional m-cryptochrome gene, linking seasonal immune regulation to the photoperiodic timer. Consistent with laboratory results, females collected under natural short-day conditions also displayed enhanced immune parameters despite increased environmental variability. Together, our findings identify photoperiod as a key regulator of immune preparedness in female insects and reveal a sex-specific anticipatory immune strategy associated with seasonal timing.

Nociception, the capacity to detect tissue-damaging stimuli such as noxious chemicals or high heat, is increasingly studied across insect orders and life stages, informing our understanding of its adaptive value and molecular mechanisms. The black soldier fly (Hermetia illucens; Diptera: Stratiomyidae) is widely recognized as the star of the growing insects as food and feed industry. Black soldier fly larvae (BSFL) are reared at high densities that, combined with their extraordinary metabolism, can generate lethal overheating on farms. Data on the thermal nocifensive capabilities of BSFL could inform our understanding of larval behaviors during overheating events (and potential welfare impacts of thermal slaughter), and provide a comparative datapoint to the well-studied vinegar fly (Drosophila melanogaster; Diptera: Drosophilidae). Accordingly, we adapted global and local thermal nociception methods from larval vinegar flies for use with BSFL. We find that global assays (akin to boiling) adapt easily to both first and sixth instar BSFL, that BSFL exhibit slightly different nocifensive behaviors than last instar vinegar fly larvae, and that BSFL have higher thresholds (thrashing begins at 39.70 {degrees}C in last instar BSFL versus 26.6 {degrees}C in D. melanogaster). In contrast, the classical local nociception assay did not adapt easily to BSFL; a modified version generated a unique, gradual pattern of increasing responsiveness to the probe in sixth instar BSFL (>95% responsiveness above 66 {degrees}C) rather than the sharp cutoff in responsiveness at 52 {degrees}C demonstrated in D. melanogaster. Altogether, these protocols open the door for standardized research on BSFL thermal nociception for fundamental and applied purposes.

Many insects enter diapause, a programmed state of developmental arrest that enables survival under adverse environmental conditions. In Drosophila melanogaster Meigen, 1830, diapause is characterized by reproductive arrest and reduced intestinal growth, accompanied by suppressed intestinal stem cell (ISC) activity. Juvenile Hormone (JH) promotes ISC proliferation under favorable conditions, but its capacity to modulate stem cell dynamics during cold-induced diapause remains unclear. Here, we investigated whether JH signaling can reactivate midgut remodeling in adult females maintained at 11. At this temperature, flies exhibited pronounced gut atrophy and elevated Phospho-histone H3 (PH3+) cell abundance, consistent with temperature-dependent G2/M phase arrest JH treatment significantly increased the proportion of Delta-positive progenitor cells in the anterior (R2) and posterior (R5) midgut regions at both 11 and 25, demonstrating that JH acts as a conserved mitogen for the ISC pool irrespective of thermal environment. A trend toward reduced PH3+ accumulation in the posterior midgut following JH treatment (p = 0.061) suggests possible facilitation of mitotic exit, though this effect did not reach statistical significance. Despite cellular-level changes, JH treatment did not restore overall gut size, indicating that the 72-84 hour exposure window was insufficient for subsequent tissue hypertrophy. Additionally, we identified a recurrent cold-induced pathology of gut distension, provisionally termed Lumen Obstruction Syndrome (LOS), which was independent of JH signaling. These findings reveal an uncoupling of JH-driven stem cell expansion from gross organ growth under diapause conditions, highlighting the selective sensitivity of the ISC compartment to endocrine signaling during environmental stress.

Insects are increasingly recognised as behaviourally complex animals, yet whether they experience pain as an affective state beyond nociception remains unresolved. Voluntary ingestion of analgesics by injured individuals has been proposed as a key but largely untested criterion for evaluating insect pain. This study tested whether Blaptica dubia cockroaches injured by wing clipping preferentially consumed an ibuprofen-sucrose solution over sucrose alone, and whether injury altered rates of abnormal behaviour when individuals were not feeding. Adult males were assigned to injured or sham-handled groups and completed 30-minute two-choice assays, with behaviour scored at 30-second intervals across analgesic, sucrose-only, and neutral zones. We also repeated the experiment with vanilla scent added as a masking agent to both analgesic and sucrose-only solutions. Injured cockroaches did not show greater preference for the analgesic solution either in the presence or absence of the vanilla masking agent. Instead, strong differences emerged between experimental conditions, with individuals in the vanilla-flavoured condition showing reduced feeding engagement overall. We therefore have no evidence that injured cockroaches actively seek out analgesics. We suggest methodological refinements are required before we can absolutely reject the possibility of analgesia preference and so the sensation of pain. In contrast, injury increased persistent abnormal behaviours, including abdominal pulsations, wing-fluttering, wound-directed grooming and body flexion. Therefore, injury produced clear behavioural disruption consistent with an internally driven aversive or discomfort-related state, highlighting both the challenges of adapting voluntary analgesic assays to insects and the welfare relevance of injury in B. dubia.

Long-distance movement in insects has crucial impacts on agriculture, human health, and biodiversity. Although it was long assumed that only large, specialist insects had the navigation capacity to support long-distance dispersal, recent studies have demonstrated that smaller insects, such as the tiny fruit fly Drosophila melanogaster, can maintain extended, straight paths while flying or walking. This raises the question of whether other Drosophila species possess the navigation capacity to support extended dispersal. Resolving this question is particularly important for Drosophila suzukii(spotted-wing drosophila), a potent pest species that causes enormous damage worldwide to ripe fruit and berries. Spotted-wing drosophila has been thought to lack a capacity for long-distance dispersal, as prior studies have estimated maximal daily dispersal distances of less than 90 m. We developed a system to continuously track the flight trajectories of magnetically tethered D. suzukii relative to a discrete, overhead LED that mimicked the sun. We found that flies maintained remarkably straight flight headings that varied unpredictably across individuals. Male and female D. suzukii exhibited a similar navigation capacity; both sexes responded to rotation of a discrete sun stimulus with compensatory turns to maintain a stable relative heading. Our results suggest that D. suzukiihas an underappreciated capacity for rapid, radial dispersal, which could exceed 250 m in 15 min. This capacity may contribute to the pest species' invasiveness and its reliable, annual re-establishment in seasonally intolerable climates. Our findings highlight the importance of developing area-wide, regional strategies to manage the impacts of D. suzukii.

Fat accumulation and use is critical for sustaining life. Most insects show a typical response to feeding where fat is accumulated when sufficient sugars and other carbohydrates are consumed. Parasitoid insects are an exception, because most species do not accumulate fat when feeding on a sugar-rich diet. Studies on fat metabolism generally measure fat content early in life without considering lipid metabolism as a dynamic process that is expected to change as life progresses. In this paper, we compared fat accumulation and use throughout the lives of adult female Drosophila melanogaster and females of 5 inbred lines of the parasitoid wasp Leptopilina heterotoma. We expected that fat accumulation would take place irrespective of teneral fat content in D. melanogaster. We found that fat D. melanogaster initially used fat reserves, while lean flies economized on fat stores. Both lean and fat flies started accumulating fat after 7 days of life, indeed showing a typical response for insects. Unlike other parasitoids, L. heterotoma populations differs in fat accumulation patterns that we expected to observe also between inbred lines. In none of the inbred lines, however, did fat accumulation take place. Our results did reveal that inbred lines differed in the rate at which fat was used mainly later during life. We further confirmed that D. melanogaster pupal size was highly correlated with adult female size for both D. melanogaster and L. heterotoma. Overall, our findings for L. heterotoma provide strong evidence that genetic background has a major impact on the rate at which fat is used over a lifetime.

In many insect species, mating is coordinated through multimodal signaling, yet less obvious channels are often overlooked. In the burying beetle Nicrophorus vespilloides, chemical communication is well-documented, but the role of substrate-borne vibrational signals (stridulations) during courtship remains unknown. We investigated whether stridulation is essential for mating success through two sets of experiments. First, we found a positive correlation between the frequency of stridulations and both the number and duration of copulation events. Second, we employed a silencing experiment to test the necessity of these signals by silencing males, females, or both partners. We found no significant differences between silenced and control groups regarding the frequency or duration of physical contact and mounting events, suggesting that stridulation is not required for mate recognition or the initiation of courtship. However, the proportion of successful copulations relative to mounting events was significantly lower when females were silenced. These results suggest that while N. vespilloides relies on a redundant multimodal system that likely utilizes chemical cues to initiate mating, vibrational signals, particularly from the female, may play a critical role in facilitating successful copulation. This study provides the first evidence for the role of stridulation in the mating behavior of N. vespilloides and highlights the potential for female-mediated vibrational signaling in burying beetle courtship.

Arboreal insects have developed various strategies to navigate their discontinuous habitats. Many insects, including leafhoppers, katydids, and praying mantises, exhibit the ability to actively leap across their leafy platforms and land on a distant substrate. This behavior is especially important for non-winged insects, including nymphal forms of winged insects, which cannot fly between these substrates. To make a targeted jump, an animal must first orient towards the target, estimate the target distance and angular location, and jump with the appropriate take-off speeds and angles to land on their intended substrate. In three-dimensional space, jumping from one point to another requires estimating distance, as well as azimuthal and elevational angles. Jumping insects such as mantises typically reorient their bodies on the substrate to align with the azimuthal direction of the target. This behavior effectively reduces the task to a two-dimensional problem, in which they must estimate only the distance to the target and its elevational angle. Many insects, including praying mantises, perform rhythmic lateral head movements called peering before performing a targeted jump. Although previous studies suggest that mechanisms such as motion parallax while peering are used for distance estimation, the full repertoire of behaviors that enable mantises to jump to arbitrarily located substrates remains unclear. We hypothesized that mantises have distinct behaviors for distance and elevation angle estimation, which enable them to independently modulate their take-off speeds and angles before jumping. To test this hypothesis, we developed behavioral assays in which mantises were placed on a launch platform and jumped to a target platform positioned at variable distances and angles. Using this apparatus, we filmed the jumps of Giant Asian mantis nymphs (Hierodula spp.) with high-speed videography and tracked body parts to quantify take-off speed and angle. Because mantis jumps are ballistic, their trajectories can be modeled as projectile motion. Our results indicate that mantises estimate target distance and elevation angle using two separate behavioral strategies: distance is assessed through peering maneuvers that generate motion parallax, whereas elevation angle is determined through visual fixation of the target accompanied by specific postural adjustments. By combining these behaviors, mantises modulate the magnitude and direction of propulsive force to achieve successful jumps.

Adult females of the two-spotted spider mite, Tetranychus urticae Koch, enter a photoperiodically induced diapause to overwinter. Diapause in T. urticae is accompanied by reproductive arrest and the orange body coloration that arises from the accumulation of astaxanthin esters. How these two traits are coordinated at the molecular level remains poorly understood. Here, we compared the proteomes of adult females reared under diapause-inducing (long-night) and non-diapause-inducing (short-night) photoperiods using liquid chromatography-tandem mass spectrometry, followed by RNA interference (RNAi) of candidate genes. The carotenoid biosynthesis enzymes phytoene desaturase (TuPDS) and lycopene cyclase/phytoene synthase (TuLCPS), both encoded by genes horizontally transferred from fungi, were more abundant in diapausing females than in non-diapausing females. RNAi of the genes encoding TuPDS and TuLCPS markedly reduced orange pigmentation as well as {beta}-carotene and astaxanthin contents, demonstrating that these enzymes are required for diapause-associated pigmentation. Our proteomic analysis further identified a single PLAT (Polycystin-1, Lipoxygenase, Alpha-toxin) domain protein, TuPLAT10, as one of the most strongly upregulated proteins in diapausing females. The PLAT domain is a lipid-binding module, suggesting a role for TuPLAT10 in lipid metabolism. In addition to the suppression of orange pigmentation, RNAi of the TuPLAT10 gene restored reproduction even under diapause-inducing conditions and selectively reduced TuPDS and TuLCPS protein levels, despite the absence of sequence similarity to their genes. We propose that TuPLAT10 acts as a lipid-allocation switch that, in response to photoperiodic information, partitions fatty acids between astaxanthin esterification and yolk lipid supply, thereby coupling reproductive arrest and carotenoid pigmentation during diapause in T. urticae.

Environmental warming is generally expected to increase metabolic demand in ectotherms. However, facultatively endothermic insects such as bumblebees regulate body temperature and may reduce thermogenic investment under warm conditions, potentially altering physiological performance and responses to climate change. We combined flow-through respirometry and infrared thermography to test how elevated ambient temperature (25 vs 35{degrees}C) affects feeding energetics and postprandial metabolism in the bumblebee Bombus terrestris. Bees maintained substantially lower thoracic temperature excess at 35{degrees}C than at 25{degrees}C, both before and during feeding. Feeding metabolic rate was also lower at 35{degrees}C and was strongly positively associated with thoracic temperature excess, indicating that feeding energetics were primarily explained by thermoregulatory state rather than ambient temperature alone. Elevated temperature reduced both the probability and energetic magnitude of specific dynamic action (SDA), including total SDA expenditure, early postprandial metabolism, and peak metabolic amplitude. In contrast, SDA duration and time to peak response showed little temperature dependence. Our results demonstrate that warming can suppress energetic expenditure in facultatively endothermic pollinators by limiting thermogenic investment and postprandial metabolic responses, potentially constraining the energetic flexibility underpinning foraging performance under climate warming.

Buzz pollination is an important behavior in which bees use vibrations to extract pollen from poricidal anthers. However, the extent to which vibration frequency influences pollen release remains unclear. Here, we quantified pollen expulsion from Solanum sisymbriifolium anthers subjected to harmonic excitation over a broad frequency range encompassing the anthers first natural frequency. We excited anthers to expel pollen and measured anther kinematics and pollen release using high-speed videography. Particle tracking enabled continuous estimation of pollen release throughout each buzzing event, allowing both initial pollen flux and total pollen released to be quantified. Pollen release depended strongly on excitation frequency. Initial pollen flux, total pollen release, and anther kinematics peaked when excitation frequency approached the anthers natural frequency. Anther tip velocity amplitude exhibited the strongest correlation with total pollen release (r = 0.755) and initial pollen flux (r = 0.898). Experimental observations were compared with nonlinear and linear statistical models of pollen release. While both models captured trends in normalized pollen flux, they overpredicted total pollen release, suggesting that adhesive interactions play important roles during extended buzzing events. These findings demonstrate that anther structural dynamics influence pollen release and suggest that vibration amplification may improve the efficiency of buzz pollination.

Feeding behavior in blood-sucking insects relies on gustatory evaluation to decide on sustained ingestion, yet quantifying this process from electromyogram (EMG) recordings is labor-intensive. Here we developed MyoRec, an automated computational framework employing machine learning to analyse EMG signals from the triatomine bug Rhodnius prolixus. Using recordings under appetitive and aversive conditions, a convolutional neural network detected ingestion events with 97.7% accuracy. Automated analysis revealed distinct feeding dynamics, with prolonged ingestion and higher pumping frequency under appetitive stimuli, compared to rapid feeding cessation under aversive stimuli. MyoRec substantially reduces analysis time while maintaining accuracy, providing a scalable tool to investigate how gustatory cues modulate feeding decisions in hematophagous insects.

A parasitic mite of honey bee brood (Tropilaelaps mercedesae), is spreading through populations of Apis mellifera honey bees in new regions and poses a major threat to honey bee health. Despite its clear threat, the biology of this mite is poorly understood, with gaps on such fundamental issues as how fast its populations can grow. This leaves the beekeeping world underprepared to plan for its arrival and management. In this study, we documented the growth of T. mercedesae populations in untreated A. mellifera colonies in Thailand and South Korea, and did the same for another parasitic mite (Varroa destructor) when possible. We found that the population growth of T. mercedesae was variable but could reach high levels (daily r of 0.010, 0.036, and 0.057), while the population growth of V. destructor (r = 0.021) matched previous estimates. Our results indicate that T. mercedesae populations can grow rapidly but they do not always attain this potential. Based on our results, humidity should be studied as a potential driver of population growth. If future work can reveal key drivers of T. mercedesae population growth, this would help predict infestations and help design management strategies that exploit the pest's biological vulnerabilities.

Phloem-feeding insects execute complex behavioral decisions to secure essential nutrients from a diet characterized by nitrogen scarcity and severe osmotic pressure due to high sucrose concentrations. We investigated the sensory mechanisms underlying these decisions in the phloem-feeding whitefly Bemisia tabaci. We demonstrate that the sweet taste receptor BtabGR1, expressed in mouthpart and gut tissues, integrates three environmental chemical cues: sucrose concentration, the presence of the essential amino acid arginine, and pH values. Arginine is a pH-dependent positive modulator of sucrose sensing, increasing receptor responses nearly fourfold under apoplast-like conditions and more than doubling the receptor responses in the gut luminal environment. Insects show a strong feeding preference for arginine-containing diets in dual-choice bioassays, with markedly higher intake when arginine is present. RNAi-mediated silencing of BtabGR1 disrupt intake regulation, leading to increased honeydew excretion. These findings suggest a putative link between arginine and the BtabGR1 receptor in regulating both feeding-site evaluation and diet ingestion. Furthermore, the ability to integrate three distinct environmental cues makes BtabGR1 one of the most complex interdependent sensory systems described for a single insect chemoreceptor.

Identifying the sensory cues that enable insects to find host plants, and understanding the neurobiology underlying their selection, provide solid foundations for developing state-of-the-art pest management strategies. Our work was aimed at identifying the main sensory cues attracting the leaf-cutting ant Acromyrmex ambiguus to alternative host plants in commercial willow plantations in the Lower Delta of the Parana River (Argentina), with a focus on native plant species. Eight plant species were selected and evaluated as potential hosts under field and laboratory conditions, allowing the establishment of a robust hierarchy of preference based on individual and collective behaviour. As a result, Senna corymbosa emerged as the most preferred species, whereas Blepharocalyx salicifolius was the least preferred. Video analyses of ant foraging in controlled indoor nests revealed a sequential decision-making process underlying plant preference and consumption. This included an initial approach driven by olfactory cues, followed by a second step involving contact-dependent cues that elicited leaf-cutting and carrying the leaf fragments to the nest. Volatile compounds and leaf cuticular components potentially involved in plant preference were identified. In addition, physicochemical analysis of both plant species - including total sugars, organic matter, polyphenols, leaf hardness, total proteins and lignin-revealed differences, particularly in polyphenol content, which may contribute to preference patterns. These findings provide insights into the sensory ecology of host preference and inform management strategies based on the reintroduction of native plants as alternative resources in willow plantations, potentially reducing pesticide use and promoting environmental sustainability. Summary statementThis work shows how leaf-cutting-ants rely on olfactory and contact cues, sequentially, for foraging decision-making of plant species. Thus, revealing how sensory cues shape their foraging decisions.

Adaptation to local environments enables species to thrive in diverse and challenging habitats. Steep elevational gradients provide a compelling natural adaptation laboratory, because abiotic conditions change progressively over short geographical differences. Given that elevation can strongly reshape physiology and behavior of insects, neuromodulatory systems offer a promising lens through which to examine elevation-specific adaptation. We challenged the hypothesis that adaptation to elevation involves octopaminergic signaling in honey bees (Apis mellifera), an important pollinator species occupying different elevations along East African mountains. We collected foragers from two distinct elevations at Mount Kenya (1,150 m and 1,900 m above sea level) and analyzed elevation-dependent changes in octopaminergic signaling. Tissue-specific analysis revealed a striking upregulation of all three octopamine {beta} receptor genes in the thoracic flight muscles and elevated octopamine brain concentrations at high elevation. Expression differences in the brain and fat body were rather modest. We subjected CRISPR/Cas9-mediated octopamine {beta}2 receptor knockouts to cold stress to study the function of octopaminergic signaling in thermoregulation. Loss of AmOAR{beta}2 reduced both the slope and amplitude of heating phases, indicating altered thermogenic dynamics. Together, these results identify the octopaminergic system as a central neuromodulatory regulator of thermogenic performance across elevations in honey bees. More broadly, our study highlights how modulation of conserved aminergic signaling pathways can shape physiological resilience to environmental gradients, pointing to a general mechanism by which insects adapt to changing thermal landscapes. Highlights- Bees from high and low elevation differ in expression of octopamine {beta} receptor genes and octopamine brain concentrations - CRISPR/Cas9-mediated octopamine receptor knockout alters thermogenic behavior - Octopaminergic signaling emerges as a key neuromodulator in thermal adaptation to elevation in honey bees Significance statementAnimals living along mountain gradients must cope with rapidly changing temperatures, yet the mechanisms enabling this adaptation remain poorly understood. We show that honey bees from higher elevations have increased brain octopamine levels and enhanced expression of octopamine receptors in heat-producing flight muscles. Using gene editing, we demonstrate that disrupting one key receptor alters how bees generate heat under cold stress. These findings identify octopamine signaling as a central regulator of thermogenesis and reveal a mechanism by which insects adjust to colder environments. More broadly, our results highlight how conserved neuromodulatory systems can fine-tune physiological performance, offering insight into how insects may respond to changing climates and expanding environmental extremes.

Electroantennography (EAG) is a valuable approach for monitoring the sensory responses of insects to insecticidal and repellent molecules and an effective tool for early screening of compounds aimed at controlling and protecting against medically important insect vectors. However, its predictive potential for repellent efficacy in triatomine vectors remains poorly explored. The objective of this study was to evaluate the EAG responses to different xenobiotics as a preliminary selection strategy for compounds with potential repellent action against triatomines. For this purpose, the antennae of adult triatomines subjected to prolonged fasting ([≥]30 days) were exposed to repellent molecules. In parallel, repellency bioassays were conducted using a live bait (Gallus gallus) and a newly designed laboratory device to validate the electroantennographic results. EAG recordings showed a significant reduction in olfactory capacity of> 60% in response to the chemical compounds IR3535 and carvone, consistent with the protection times observed in the repellency tests (135.6 {+/-} 43.29 min and 108 {+/-} 26.33 min, respectively). In conclusion, the compounds with the highest repellent activity were clearly discriminated by the insects olfactory system, a finding corroborated by the decrease in electrical signals recorded in the EAG bioassays.

The tick Ixodes ricinus is the main vector of human and animal pathogens in Europe. Despite its importance in epidemiology and medicine, our understanding of physiological mechanisms controlling blood feeding, osmoregulation, or development are still limited. Here, we identify novel neuropeptide invertebrate parathyroid hormone-like peptide (iPTH) and its two receptors - PTHR1 and PTHR2 in I. ricinus. Functional aequorin-based assay confirmed specific activation of both receptors by iPTH. Using RT-qPCR we detected the PTHR1 transcript in the synganglion, while increased expression levels of PTHR2 were found in the salivary glands, hindgut and female gonads. RNA-mediated knockdown of iPTH receptors in nymphs resulted in delayed blood feeding, and a high incidence of defects in adult ecdysis. Consistent with observed phenotypes, iPTH is expressed in multiple neurons of the synganglion which project arborizing axons to the salivary glands, rectal sack and skeletal muscles. iPTH was colocalized with orcokinin-immunoreactivity (OK-IR) in all neurons that innervate these peripheral tissues. iPTH is further colocalized with tachykinin (TK) in Pd1DL1 neurons, suggesting coordinated action with other neuropeptides. Our findings indicate that iPTH signaling is required for normal feeding, development and successful ecdysis.

The black soldier fly (Hermetia illucens, Diptera) undergoes an atypical metamorphic program in which a distinct non-feeding prepupal instar precedes pupation, but its developmental and molecular basis remains poorly understood. Here, we investigated this unusual metamorphic program through integrated developmental and RNAi-based analyses. Postembryonic staging confirmed that the 6th instar feeding larva molts into a non-feeding 7th instar prepupa, whose hardened cuticle subsequently serves as the puparium during intra-puparial development. Expression profiles of four key metamorphic genes revealed a stage-specific Chinmo/Kr-h1-Br-c-E93 regulatory shift corresponding to larval, prepupal, and pupal/adult development, with sustained Br-c expression defining the 7th instar prepupal stage. RNAi-mediated knockdown of Kr-h1 and/or Chinmo induced precocious larval-prepupal metamorphosis, supporting their roles in larval stage maintenance, whereas depletion of Br-c or E93 disrupted prepupal-pupal transition and adult differentiation, respectively, consistent with their functions as pupal and adult specifiers. These results support generally conserved functions of the metamorphic gene network while indicating the absence of a repressive effect of Br-c on E93 in H. illucens prepupae. Together, these findings establish the 7th instar prepupa as an independently regulated transitional stage, providing insight into how metamorphic programs are reorganized to diversify life-history strategies in holometabolous insects.

Most organisms have evolved mechanisms to reduce the negative effects of toxins in their diet. Some animals that are toxin specialists, such as dendrobatid poison frogs, have amino acid substitutions in proteins targeted by the toxins that prevent or limit the toxins ability to bind and exert its bioactive effects. The Phantasmal poison frog, Epipedobates tricolor, has amino acid substitutions in its neuronal nicotinic acetylcholine receptors that were previously shown to provide resistance to the highly potent neurotoxin epibatidine in vitro. However, it is unclear whether E. tricolor resists the physiological effects of epibatidine in vivo. To investigate this, we examined the effects of epibatidine exposure on the locomotion and behavior of E. tricolor. We performed whole-animal performance assays and behavioral evaluations at multiple time points following the administration of high yet biologically relevant levels of epibatidine. These assays were followed by alkaloid quantification to track chemical concentrations in the skin. Epipedobates tricolor exhibited similar locomotor performance and behavior at both epibatidine doses compared to controls and regardless of the quantity of alkaloid accumulated into the skin. However, we observed an impact of low-percentage ethanol solutions on behavior when compared to water controls, as well as general impacts of handling stress (regardless of the administered solution type), which should be considered in future experimental designs. Overall, we demonstrate that E. tricolor likely avoids a physiological fitness trade-off between toxin ingestion and the defensive benefits of epibatidine sequestration. Our study suggests that the ability to ingest toxins involves multi-faceted resistance mechanisms. Highlights- Whole-animal performance assay shows no impact of epibatidine on frog behavior - Epipedobates tricolor is 20-5000 times more resistant to epibatidine than mice - Assays demonstrate that frogs are sensitive to 6% ethanol and handling stress